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Whole Plant Response of Chrysanthemum to Paclobutrazol, Chlormequat Chloride, and (s)-Abscisic Acid as a Function of Exp...

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Permanent Link: http://ufdc.ufl.edu/UFE0022498/00001

Material Information

Title: Whole Plant Response of Chrysanthemum to Paclobutrazol, Chlormequat Chloride, and (s)-Abscisic Acid as a Function of Exposure Time Using a Split-Root System
Physical Description: 1 online resource (61 p.)
Language: english
Creator: Boldt, Jessica
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: abscisic, acid, chloride, chlormequat, chrysanthemum, growth, paclobutrazol, plant, regulators, split
Environmental Horticulture -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Paclobutrazol and chlormequat chloride inhibit production of gibberellins and internode elongation whereas (S)-abscisic acid is used as an anti-transpirant to increase postharvest longevity while maintaining aesthetic quality of plants exposed to drought. Little is understood of the timing of chemical movement into the plant following drench applications to peat based media. The split-root system was developed to evaluate chemical uptake in a whole plant system, similar to commercial application situations. The objective of this study was to evaluate uptake of paclobutrazol, chlormequat chloride, and (S)-abscisic acid applied as a media drench and determine the critical uptake period. Roots of chrysanthemum (Dendranthema x grandiflora) ?Snowmass? were separated and grown in two adjoining compartments of a cell pack. The chemical being evaluated was applied to one-half of the root system, which was excised at prescribed time intervals to terminate the plant?s exposure to the chemical. Uptake was determined as a function of plant response. Paclobutrazol uptake was slow and continual, reaching a plateau at 15.85 days with elongation 44% of the control. Chlormequat chloride uptake was rapid, reaching a plateau at 6.78 hours with elongation 55% of the control. (S)-abscisic acid uptake was rapid as all exposure intervals between 7.5 min and 4 h had transpiration of 0.4 microgram/sq. cm/sec, compared to 9.7 microgram/sq. cm/sec for the uncut water control, measured at 4 h after treatment (HAT). At 24 and 48 HAT, transpiration of the uncut control was 14.0 and 13.1, the 7.5 min exposure interval was 4.4 and 5.5, and the 1 h exposure interval was 2.5 and 3.6 microgram/sq. cm/sec, respectively. Differences in efficacy between exposure intervals and across time were probably due to metabolism. Uptake differences were observed with the chemicals in this study. Paclobutrazol uptake is slow, probably due to adsorption and desorption reactions between the chemical and organic matter in the media. Chlormequat chloride and (S)-ABA are taken up rapidly and remain in solution. Uptake of these chemicals is by mass flow with the transpiration stream and could possibly be affected by factors affecting transpiration, such as light, temperature, or humidity, at or shortly after application.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jessica Boldt.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Barrett, James E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022498:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022498/00001

Material Information

Title: Whole Plant Response of Chrysanthemum to Paclobutrazol, Chlormequat Chloride, and (s)-Abscisic Acid as a Function of Exposure Time Using a Split-Root System
Physical Description: 1 online resource (61 p.)
Language: english
Creator: Boldt, Jessica
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: abscisic, acid, chloride, chlormequat, chrysanthemum, growth, paclobutrazol, plant, regulators, split
Environmental Horticulture -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Paclobutrazol and chlormequat chloride inhibit production of gibberellins and internode elongation whereas (S)-abscisic acid is used as an anti-transpirant to increase postharvest longevity while maintaining aesthetic quality of plants exposed to drought. Little is understood of the timing of chemical movement into the plant following drench applications to peat based media. The split-root system was developed to evaluate chemical uptake in a whole plant system, similar to commercial application situations. The objective of this study was to evaluate uptake of paclobutrazol, chlormequat chloride, and (S)-abscisic acid applied as a media drench and determine the critical uptake period. Roots of chrysanthemum (Dendranthema x grandiflora) ?Snowmass? were separated and grown in two adjoining compartments of a cell pack. The chemical being evaluated was applied to one-half of the root system, which was excised at prescribed time intervals to terminate the plant?s exposure to the chemical. Uptake was determined as a function of plant response. Paclobutrazol uptake was slow and continual, reaching a plateau at 15.85 days with elongation 44% of the control. Chlormequat chloride uptake was rapid, reaching a plateau at 6.78 hours with elongation 55% of the control. (S)-abscisic acid uptake was rapid as all exposure intervals between 7.5 min and 4 h had transpiration of 0.4 microgram/sq. cm/sec, compared to 9.7 microgram/sq. cm/sec for the uncut water control, measured at 4 h after treatment (HAT). At 24 and 48 HAT, transpiration of the uncut control was 14.0 and 13.1, the 7.5 min exposure interval was 4.4 and 5.5, and the 1 h exposure interval was 2.5 and 3.6 microgram/sq. cm/sec, respectively. Differences in efficacy between exposure intervals and across time were probably due to metabolism. Uptake differences were observed with the chemicals in this study. Paclobutrazol uptake is slow, probably due to adsorption and desorption reactions between the chemical and organic matter in the media. Chlormequat chloride and (S)-ABA are taken up rapidly and remain in solution. Uptake of these chemicals is by mass flow with the transpiration stream and could possibly be affected by factors affecting transpiration, such as light, temperature, or humidity, at or shortly after application.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jessica Boldt.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Barrett, James E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022498:00001


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1 WHOLE PLANT REPONSE OF CHRYSA NTHEMUM TO PACLOBUTRAZOL, CHLORMEQUAT CHLORIDE, AND (S)-ABS CISIC ACID AS A FUNCTION OF EXPOSURE TIME USING A SPLIT-ROOT SYSTEM By JESSICA LYNN BOLDT A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Jessica Lynn Boldt

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3 To Mom and Dad for all their love and support.

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4 ACKNOWLEDGMENTS I would like to thank m y major professor, Dr. Jim Barrett, for being a great professor, mentor, and friend. My graduate experience and research ability has been greatly enhanced by all the opportunities you have exposed me to de veloping a research program, being a teaching assistant, giving class lectures, performing va riety trialing, attending industry events, and presenting research at academic conferences. Th ank you also for allowing me to enjoy growing poinsettias and not making it my research crop! I would like to thank my committee members, Drs. Jamie Gibson, Paul Fisher, and Greg MacDonald for their research guidan ce and help in manuscript review. I would like to thank Carolyn Bartuska for he r assistance in statistical analysis and Bob Weidman for taking care of my plants when I was away from the greenhouse. Most of all, I would like to thank my parents and my twin sister Jennifer for all their support and encouragement during this endeavor. You always kept me focused and encouraged, even when things did not always go as planned.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........6 LIST OF FIGURES.........................................................................................................................7 ABSTRACT.....................................................................................................................................8 CHAP TER 1 INTRODUCTION..................................................................................................................10 Introduction................................................................................................................... ..........10 Paclobutrazol..........................................................................................................................12 Chlormequat Chloride............................................................................................................15 Abscisic Acid..........................................................................................................................17 Introduction................................................................................................................... ..17 Synthesis and Metabolism...............................................................................................20 Movement........................................................................................................................22 Signaling Sequence.........................................................................................................23 Role in Stomatal Closure.................................................................................................24 Abscisic Acid as a Chemical Signal................................................................................ 25 Agricultural Importance.................................................................................................. 27 2 WHOLE PLANT RESPONSE OF CHRYSANTHEMUM TO PACLOBUTRAZOL AND CHL ORMEQUAT CHLORIDE AS A FUNCTION OF EXPOSURE TIME USING A SPLIT-ROOT SYSTEM........................................................................................ 30 Introduction................................................................................................................... ..........30 Materials and Methods...........................................................................................................30 Results and Discussion......................................................................................................... ..32 3 WHOLE PLANT RESPONSE OF CHRYSANTHEMUM TO (S)-ABSCISIC ACID AS A FUNCTION OF EXPOSURE TI ME USING A SPLI T-ROOT SYSTEM..................39 Introduction................................................................................................................... ..........39 Materials and Methods...........................................................................................................40 Results and Discussion......................................................................................................... ..41 4 CONCLUSION..................................................................................................................... ..49 WORKS CITED.................................................................................................................... ........53 BIOGRAPHICAL SKETCH.........................................................................................................61

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6 LIST OF TABLES Table page 1-1. Summary of studies appl ying exogenous ABA and evaluating effects on stom atal closure................................................................................................................................29 2-1 Plant elongation response following applicati on of water to one half of root system ....... 38 2-2 Plant elongation response to exposure tim e following chlorm equat chloride media drench to one half of root system....................................................................................... 38

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7 LIST OF FIGURES Figure page 2-1. Paclobutrazol linear-plateau model fo r chrysanthem um elongation response to exposure time following media drench to one half of root system.................................... 36 2-2. Chlormequat chloride linear-plateau model for chrysanthem um elongation response to exposure time following media drench to one half of root system................................ 37 3-1. Effects of (S)-ABA exposure interval on transpiration rate at 4 hours after application. ................................................................................................................... ......46 3-2. Effects of (S)-ABA exposur e interval on transp iration rate at 24, 48, and 72 h after application .................................................................................................................... ......47 3-3. Effects of (S)-ABA exposur e interval on transp iration rate at 4, 24, and 48 h after application. ................................................................................................................... ......48

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8 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science WHOLE PLANT RESPONSE OF CHRYSANTHEMUM TO PACLOBUTRAZOL, CHLORMEQUAT CHLORIDE, AND (S)-ABS CISIC ACID AS A FUNCTION OF EXPOSURE TIME USING A SPLIT-ROOT SYSTEM By Jessica Lynn Boldt August 2008 Chair: James Barrett Major: Horticultural Science Paclobutrazol and chlormequat chloride inhi bit production of gibbere llins and internode elongation whereas (S)-abscisic acid is used as an anti-transpirant to increase postharvest longevity while maintaining aesthetic quality of pl ants exposed to drought. Little is understood of the timing of chemical movement into the plan t following drench applications to peat based media. The split-root system was developed to evaluate chemical uptake in a whole plant system, similar to commercial app lication situations. The objective of this study was to evaluate uptake of paclobutrazol, chlormequat chloride, an d (S)-abscisic acid applie d as a media drench and determine the crit ical uptake period. Roots of chrysanthemum ( Dendranthema x grandiflora ) Snowmass were separated and grown in two adjoining compartmen ts of a cell pack. The chemical being evaluated was applied to one-half of the root system, which was excised at prescribed time intervals to terminate the plants exposure to the chemical. Uptake was de termined as a function of plant response. Paclobutrazol uptake was slow and continua l, reaching a plateau at 15.85 days with elongation 44% of the control. Chlormequat chlo ride uptake was rapid, reaching a plateau at 6.78 hours with elongation 55% of the control. (S)-abscisic acid uptake was rapid as all

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9 exposure intervals between 7.5 min and 4 h ha d transpiration of 0.4 microgram/sq. cm/sec, compared to 9.7 microgram/sq. cm/sec for the un cut water control, m easured at 4 h after treatment (HAT). At 24 and 48 HAT, transpir ation of the uncut c ontrol was 14.0 and 13.1, the 7.5 min exposure interval was 4.4 and 5.5, and the 1 h exposure interval was 2.5 and 3.6 microgram/sq. cm/sec, respectively. Differences in efficacy between exposure intervals and across time were probably due to metabolism. Uptake differences were observed with the chem icals in this study. Pa clobutrazol uptake is slow, probably due to adsorption and desorption reactions between the chemical and organic matter in the media. Chlormequat chloride a nd (S)-ABA are taken up rapidly and remain in solution. Uptake of these chemicals is by mass flow with the transpiration stream and could possibly be affected by factors aff ecting transpiration, such as light temperature, or humidity, at or shortly after application.

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10 CHAPTER 1 INTRODUCTION Introduction Plant growth regulators (PGRs) are orga nic compounds, which in sm all amounts, are applied to promote, inhibit, or otherwise modi fy any physiological plant process (Tukey et al., 1953). This general class can be further divide d into plant hormones, or phytohormones, which are naturally occurring compounds produced by a plant that move fr om a site of synthesis to a site of action (Tukey et al., 1953) and plant growth retardants which are synthetic compounds applied to control plant size without obvious phytotoxicity (D avis and Curry, 1991). Plant growth retardants have been widely researche d, developed, and used commercially for the past 40 to 50 years to manipulate plant shape, form, and overall crop quality for agricultural and horticultural purposes. For ornamental crops, gr owth regulators are important for producing compact or appropriately-sized plants, maintaining quality prior to sale, promoting shelf-life, and improving aesthetic quality (Arteca, 1996). To be commercially desirable, growth regulators must provide consistent result s across a reasonable range of environmental and cultural conditions that could occur duri ng production (Davis and Curry, 1991). Typical application methods for plant grow th regulators are fo liar sprays or media drenches, but also include media sprays, bulb and seed soaks, and cutting and liner dips. Each method has advantages and disadva ntages that must be consider ed so the proper application technique is used. Sprays t ypically are easier and quicker to apply, but often have less uniformity due to transport resistance across the leaf surface or re duced coverage from overlapping plant canopies, especially in multi-pl ant containers. Higher concentrations are applied and phytotoxicity is more lik ely to occur. Drenches are more labor intensive than sprays, but produce longer-lasting and more uniform results. Another advantage is greater control on

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11 typically insensitive crops or crops requiring greater si ze reduction, even though lower concentrations are applied (G ent and McAvoy, 2000). Newer tec hniques gaining popularity due to uniform, early growth control are bulb soaks and liner dips. Plant growth regulators are extremely importa nt to the horticulture industry, both during production and at retail. Even though research has been conducted to determine uptake, translocation, metabolism, and mode of action, basic and applied rese arch questions still remain. It is known that a threshold concentration of a plant growth regulator must be present for promotion or inhibition of in tended biological activity (Birec ka, 1967; Lever, 1986) and that plant growth regulators are pass ively taken up by the root syst em from media drenches or submersion in solution (Lever, 1986). Howeve r, it is unknown how much chemical exposure time is required to achieve maximum efficacy a nd which portion of the uptake period is most critical for efficacy. Research i nvestigating the uptake pattern of growth regulators relative to efficacy could provide information to answer applied questions, including the importance of environmental conditions at the time of applicatio n, differences in plant response to application method, and the recommendation that media applicat ions should be applied in the morning to actively growing plants to a void potential proble ms with phytotoxicity (Armitage, 1994). A split-root system using chrysanthemum ( Dendranthema x grandiflorum ) Snowmass was developed to determine the up take of media-applied plant grow th regulators as a function of plant response. Three growth regulators were inve stigated two plant grow th retardants and one plant growth hormone. Chlormequa t chloride and paclobut razol are important to the horticulture industry and commonly used as media drenches to control plant size. Abscisic acid is currently under development for commercial use to regulate water loss of horticulture crops at retail and subsequently improve aesthetic quality and increase shelf-life. The following sections provide

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12 information about the discovery, chemistry, transport, accumulation, metabolism, and importance of these three plant growth regulators. Paclobutrazol Paclobutrazol ([2RS, 3RS]-1-[4-chlorophenyl]-4, 4-dim ethyl-2-[1, 2, 4-triazol-1-yl]pentan3-ol) is an important plant gr owth retardant that reduces pl ant growth and increases the commercial and aesthetic value of many floriculture and ornamental crops. Triazole growth regulator activity was discovered during screenin g programs for fungicidal activity (Davis et al., 1988) and paclobutrazol was disc overed in 1980 by ICI Plant Prot ection Division (Goulston and Shearing, 1985). The molecule has a ring struct ure containing three nitrogen atoms (Davis and Curry, 1991) and consists of two enantiomers. The (2R, 3R) enantiomer provides fungicidal activity while the (2S, 3S) enantiomer provides growth regulating activity (Sugavanam, 1984). It was identified in initial resear ch as PP333 and is commercially available under the trade names of Bonzi, Downsize, Paczol, Piccolo, Clipper, Cultar, and Parlay (L ever, 1986; Barrett, 2006). Paclobutrazol is a potent regula tor of gibberellin biosynthesis and inhibits the oxidation of kaurene to kaurenoic acid. Speci fically, it interacts with kaur ene oxidase, a cytochrome P-450 oxidase, and inhibits the microsomal oxidation of kaurene, kaurenal, a nd kaurenol (Hedden and Graebe, 1985). Reduced levels of gibberellins lead to a decrea se in cell division and elongation at the apical meristem of the shoot, but has little effect on the production of leaves or root growth (Giafagna, 1995). Paclobutrazol must be translo cated to the meristematic region and maintain a threshold concentration for efficacy (Lever, 1986). It is often referred to as an anti-gibberellin because physiological treatment effects can be reversed by the application of GA (Cox, 1991). However, this term is misleading because pacl obutrazol does not block the activity of existing endogenous or applied exogenous GA3 (Lever, 1986) by competing for the same active site. Instead, paclobutrazol should be referred to as a gibberellin inhibitor.

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13 Transport of paclobutrazol occurs passively in the xylem (Barrett and Bartuska, 1982; Wang et al., 1986; Early and Ma rtin, 1988), with little to no movement in the phloem (Richardson and Quinlan, 1986). It was proposed th at transpiration was required to draw the chemical through the xylem to the meristematic regions (Lever, 1986) and confirmed by Early and Martin (1988), who demonstrated using 14C-paclobutrazol that the pattern of radioactivity followed the pathway of normal water movement. A previous study indicated that paclobutrazol uptake from solution and movement within tissue was rapid, with significant levels of labeled material detected within 12 hour s of treatment (Early, 1986). M ovement of paclobutrazol in the plant is acropetal, with no m ovement out of mature leaves (R ichardson and Quinlan, 1986; Early and Martin, 1988). Accumulation with in a plant is primarily in r oot and leaf tissue, with one study determining ~80% of labele d material accumulated in basa l and midsection leaves (Early and Martin 1988). Only a small portion of applie d paclobutrazol actually reaches the site of action. Metabolism is generally thought to occur very slowly within plant ti ssue, but there have been differing reports. Early and Martin (1988) reported that breakdown of paclobutrazol ranged from 32 to 58.5%, with most degradation occurr ing in leaf tissue, as only 7.8 to 12.2% of 14C activity remained as paclobutrazol nine days afte r treatment. However, Sterrett (1985) reported in apple that 85% of 14C activity in shoot tissue remained as paclobutrazol 27 days after injection. Degradation of paclobutrazol occurs in soil under aerobic conditions and mesophilic temperatures due to microbial activ ity (Jackson et al., 1996). Eight Pseudomonas and one Alcaligenes have been identified as having the capacity to degrade paclobutrazol. Degradation is temperature dependent, with a test in aqueous suspensions determ ining that it takes 4.5 years at

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14 25C and 2.5 years at 60C. With the addition of Pseudomonas degradation was very rapid and took 13 days (Jackson, Line and Hasan 1996). In field situations, paclobu trazol has a half-life ranging from 3 to12 months (L ever 1986) or 12 to 18 months although some have reported persistence as long as 3 years (Jacyna a nd Dodds, 1995). Some commercial greenhouse operations have had issues dealing with chemical residues. Paclobutrazol is a wide spectrum growth retardant that shows a response over many species, including some that were insensitive to ot her classes of plant growth retardants. Plants treated with paclobutrazol typical ly have shorter internodes and thicker green leaves. Response is dependent on many factors, including species and/or cultivar sensitivity, cultural and environmental conditions, media composition, irrigation method, application method, and application dose. Paclobutrazol has a low water solubility of 30 to 35 ppm and is a non-polar molecule. Binding to soil components is related to organi c matter content, clay content, and cation exchange capacity (Davis, 1988) while efficacy in commercial potting media is related to bark percent and composition (Million et al., 1998). A hydrophobi c attraction exists between the nonpolar portion of the paclobutrazo l molecules and the waxy layers of the bark, creating a reversible adsorption reaction (B arrett, 1982). These binding reac tions are thought to occur very rapidly because no loss of efficacy was observed due to leaching during a pplication (Million et al., 1999) or irrigation as soon as 1 hour after ap plication (Barrett et al., 1987). Over time, paclobutrazol desorbs from the bark and is availa ble for uptake by the root s (Barrett, 1982). Adsorption reactions generally take place in the upper levels of the soil and media, above the root zone (Barrett, 1982), and were confirme d using a bioassay. Over time, there was a slow re-distribution to the middle and lower layers of the media (Million et al., 1999). Uptake and

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15 efficacy of paclobutrazol is dependent on root prox imity (Davis, 1988). It has been proposed in certain situations that paclobutrazol could effectively work as a slow-release growth retardant due to its slow desorption from soil compone nts and prolonged efficacy (Jacyna and Dodds, 1995). Paclobutrazol does not pose a leaching hazard, but moves slowly through the soil and media profile over time. Chemical activity in commercial media is infl uenced by the amount and properties of bark present. Bark particle size is important, with activity reduced more in the fine (<2 mm) fraction of fresh and composted bark samples than in medium (2-4 mm) or coarse (>4 mm) fractions (Million et al., 1998). Type of bark also influences efficacy, with a 4-, 5-, and 10-fold higher concentration of paclobutrazol re quired for old composted bark, fresh pine bark, and composted pine bark to achieve si milar height reduction as Sphagnum peat or coir based media (Million et al., 1998). The reduction in chemical efficacy ha s been proposed to be related to component surface area and density (Million et al., 1998). Chlormequat Chloride Chlorm equat chloride was discovered during a screening program of quaternary ammonium compounds for growth re tardant activity and first desc ribed by Tolbert (1960a). It has been used extensively since the 1960s to pr event lodging in grain and cereal crops and for growth control of potted greenhous e crops, especially poinsettia ( Euphorbia pulcherrima ), chrysanthemum (Dendranthema x grandiflora ), azalea ( Rhododendron sp.), geranium ( Pelargonium hortorum ), and hibiscus ( Hibiscus rosa-sinense ). Chlormequat chloride is commercially available under the trade name Cycocel. Chlormequat chloride, also known as chlorocholine chloride, is an onium growth retardant containing a quaternary ammonium group (Gent and McAvoy, 2000). It has a chemical name of (2-chloroethyl) trimethyla mmonium chloride and a chemical formula of C5H13Cl2N arranged as

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16 Cl2CH2CH2N(CH3)3 (Tolbert, 1960a). It is a choline derivative contai ning a substituted Cl for a hydroxyl group (Tolbert, 1961; Cathey, 1964). Chlorme quat chloride inhibits the cyclization of geranylgeranyl pyrophosphate to copallyl pyrophosphate in the gi bberellin biosynthesis pathway (Rademacher, 2000). Chlormequat chloride is highly mobile in both xylem and phloem tissue (Lord, 1981; Kust, 1986) and rapidly absorbed and tr anslocated. It is highly wate r soluble (Cathey and Stuart, 1961) and passively absorbed by all plant tissues, allowing it to be effectively applied as a spray or drench (Tolbert, 1960b). Howeve r, studies with winter barley indicate that applications were more effectively taken up by the roots than the le aves (Belzile et al., 1972). In one study with wheat (Dekhuijzen and Vonk, 1974) uptake during the first six hours by the roots resulted in 20% growth inhibition two weeks la ter. Chlormequat chloride preferentially accumulates in the meristematic regions (Belzile et al., 1972), ne wly expanding leaves (In trieri and Ryugo, 1974), and young tissues and organs (Birecka, 1967) with so me re-distribution to root tissue (Intrieri and Ryugo, 1974). Studies have estimated that 25 to 33% of applied chlormequat chloride in plant tissue is present in th e upper portion of the plant (B irecka ,1967; Dekhuijzen and Vonk, 1972). Metabolism of chlormequat chlo ride is thought to occur rapi dly, but differing results have been reported. Early reports suggested chlorm equat chloride was not broken down in plant tissues (Birecka, 1967; Blinn, 1967) contributing to the persiste nce of growth retardation (Cathey, 1964). However, further studies showed that chlormequat chloride was broken down to choline by substitution of the chlorine ion with a hydroxyl group (Belzile et al., 1972; El-Fouly and Jung, 1969; Schneider, 1967). One study hypothe sized that reduced chlormequat chloride efficacy and duration of control compared to other plant growth retardants discovered around the

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17 same time was due to rapid metabolism to choline, a compound with li ttle growth regulator activity (Schneider, 1967). Further metabolism results in th e subsequent conversion of choline to betaine, glycine, and serine, which are inco rporated into the protein fraction of the plant (Stephan and Schutte, 1970; Dekhuijzen and Vonk, 1974). Metabolism of chlormequat chloride is rapid, with Jung and El-Fouly (1966) reporting co mplete breakdown to choline within 10 days. Other researchers determined the biological halflife to be 13 days (Mooney and Pasarela, 1967) and 25 days (Bier and Dedek, 1970) and the e ffective growth retarding period to be approximately 20 days (Intrieri and Ryugo, 1974) Chlormequat chloride is also rapidly metabolized by soil microorganisms (Blinn, 1967 ; El-Fouly and Jung, 1969) or broken down by steam sterilization (Cathey, 1964) and does not persist from one crop to the next (Cathey and Stuart, 1961). Application of chlormequat chlo ride to crops results in plan ts with shorter internodes and thicker, darker green leaves (Tolbert, 1960a, 1960b) A threshold concentration is required for growth inhibition (Birecka, 1967), with reports of low concentrations actually promoting stem elongation (Tolbert, 1961; Halevy and Wittwer, 1965) Improper application or excessively high concentrations result in severe marginal leaf chlorosis (Armitage, 1994) or chlorotic spotting. Abscisic Acid Introduction Abscisic acid was first identified as a subs tance controlling plant physiological processes in the 1960s by three research groups. A group at the University of California, Davis with Carns and Addicott investigated substanc es controlling abscission in cotton (Gossypium hirsutum ). An initial substance was identified in the 1950s and nam ed abscisin, but it was never isolated or quantified (Addicott and Lyon, 1969). A second and more active compound isolated from young cotton bolls shown to promote leaf abscission was named abscisin II, characterized (Ohkuma

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18 et al., 1963), structure determined (Ohkuma et al ., 1965), and confirmed by synthesis (Cornforth et al., 1965). A second group at Milstead La boratory, University of Wales with Wareing investigated a compound isolat ed from sycamore leaves ( Acer pseudoplatanus ) that induced resting bud formation (Robinson and Wareing, 1964) and called it dormin. Isolation of the compound (Cornforth et al., 1965) led to the discovery that abscisin II and dormin was the same compound and confirmed the chemical struct ure. A third research group at Wye College, University of London with Rothwell and Wain (1964) continued preliminary studies of Van Steveninck with a compound pres ent in the fruit of lupin ( Lupinus luteus var. Weiko II) that induced flower drop. It was identified as be ing identical to absci sin II and dormin. To reduce confusion associated with mu ltiple names for the same compound, it was renamed abscisic acid, abbreviated ABA, by the i nvolved researchers and pr esented at the Sixth International Conference in Plant Growth Subs tances (Ottowa, July 24-28, 1967) (Addicott et al., 1968). Reasons for the proposed name included: (a) indication of the compounds chemical nature, (b) facilitated na ming of derivatives, and (c) close enough to the original name to avoid confusion. Confirmation of the name cha nge was published in 1968 (Addicott et al.). Abscisic acid has a chemical formula of C15H20O4, a molecular weight of 264 mass units (Ohkuma et al., 1963), and a chemical name of 3-methyl-5-(1-hydroxy-4-oxo-2,6,6trimethyl-2-cyclohexen-1-yl)cis trans -2,4-pentadienoic acid (Addico tt et al., 1969). Synthesis of ABA results in two isomers (or enantiomorphs ), designated as ()-ABA or (RS)-ABA, with (+)-ABA or (S)-ABA as the naturally occurr ing form. ABA can also isomerize to a trans configuration as a 1:1 equilibrium mixture when exposed to light, but the compound is biologically inactive (Mousseron-Canet, 1966, cited by Milborrow, 1970).

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19 Early research indicated both (+ )and (-)-ABA have biologic al activity, making it the first optically active plant hormone (Milborrow, 1970). Cornforth et al. (1965) reported synthetic racemic ABA had one-half the inhibitory activity of the natural form as determined by bioassay., but Milborrow reported both forms of ABA were equally active in inhibiting coleoptile growth of dissected wheat embryos. Sondheimer et al (1971) determined that germination, shoot growth, and root growth of barley ( Hordeum vulgare ) responded to both fo rms of ABA, but (S)ABA was more effective. Howeve r, research soon demonstrated that only (+)-ABA was capable of controlling stomatal function. Kriedemann et al. (1972) showed that ()-ABA was approximately one-half as effective at the same concentration as (+)-ABA, but doubling the concentration of ()-ABA provided results equal to initial (+)-AB A concentration. Results were confirmed by Cummins and Sondheimer (1973). Abscisic acid regulates many plant physio logical processes, including abscission, dormancy, germination, growth, root geotropism, and stomatal function. It is now classified as a naturally occurring plant hormone based on meeting the following requirement s: (a) it occurs in numerous plant species and plant tissues, (b) it is present endoge nously in small quantities, and (c) it moves from the site of synthesis to th e site of action (Addicott and Lyon, 1969; Wareing, 1978). When initially discovered, multiple patents were issued for agriculture use (Addicott and Lyon, 1969) but ABA is not widely used due to ra pid metabolism in plant tissue, high cost of production due to expensive starting materials and low yield potential, and instability in UV light (Addicott and Lyon, 1969; Milborrow, 1969; Gian fagna, 1995). Much research has focused on the role of ABA in regulation of plant water status in response to environmental stress, mainly drought stress, and the possibility of use as an an ti-transpirant for efficient water usage and crop protection.

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20 Synthesis and Metabolism Abscisic acid synthesis has been studied exte nsively, but the com plete pathway is not fully understood. It is a sesquiterpenoid C15 compound composed of three isoprene residues and can be synthesized by two pathways, either directly from a C15 precursor or indirectly from the cleavage of a C40 carotenoid (Milborrow, 1969). Both pathways have two compounds in common, starting with mevalonic acid (MVA) whic h is converted to fa rnesyl pyrophosphate (FPP) (Walton and Li, 1995). At this point, the direct pathway l eads to abscisic acid while the indirect pathway continues s ynthesis to violaxanthin, a C40 carotenoid compound, with ABA formed from enzymatic cleavage. Studies using labeled ABA were inc onclusive due to poor incorporation of radioactive precursors, such as MVA and CO2 (Walton and Li, 1995). Further research has identified the primary synthesis pathway as originating from a C40 carotenoid. Work with several corn ( Zea mays) mutants that lack the ability to synthesize carotenoids and application of carotenoid synthesis inhibitors produced plants that had a diminished ability to accumulate ABA (Walton and Li, 1995). Additional strong evidence for the direct pathway arose from a feeding st udy by Creelman and Zeevaart (1984) using labeled 18O. The proposed pathway of synthesis is tr ans violaxanthin, 9-cis-neoxanthin, xanthoxin, ABA aldehyde, and finally ABA (Li and Walton, 1990). Abscisic acid synthesis occurs in response to environmental stress conditions, especially water stress. ABA concentration in leaf tissue has been shown to increase 10 to 50 fold within 4 to 8 hours of water stress, with the dramatic rise in ABA levels primarily due to increased rate of biosynthesis (Walton and Li, 1995). This rapid response protects the plant from both immediate and long term damage due to excessive water lo ss under adverse conditions. As soon as plant water status returns to normal, ABA is rapidly metabolized so normal functioning resumes. It

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21 has been proposed that the mechanism for sensing plant water status and regulating the increase in ABA synthesis or metabolism is leaf turgor pressure (Pierce and Raschke, 1980). Early research was successful in identifying the main substances produced as ABA was metabolized; however, rate of metabolism a nd metabolites produced differs between ABA enantiomers. Researchers conclude that almost any change in ABA configuration results in inactivation and limited to no biol ogical activity. Studies with is olated metabolites from bean axes ( Phaseolus vulgaris ), designated as M-1 and M-2 (Sond heimer et al., 1971), indicated one metabolite was converted to the other (Walton and Sondheimer, 1971), later identified as phaseic acid (PA) and dihydrophaseic aci d (DPA) (Milborrow, 1974). S ite of ABA metabolism was hypothesized to occur in the cy toplasm (Milborrow, 1979) and c onfirmed by experimentation in spinach leaves ( Spinacia oleracea ) (Hartung et al., 1980). Abscisic acid metabolism is rapid, especia lly under stress conditi ons, but reported rates vary extensively between hours and days. Two main classes of studies have been conducted, with one group using exogenously applied labeled ABA and one group observing endogenous ABA and metabolite levels during stress cycl es and recovery periods. In spinach (S pinicia oleracea ), 32% and 64% of leaf-applied 14C-ABA was metabolized after 8 and 24 hours, respectively (Hartung et al ., 1980). In grape vine (Vitis vinifera ) leaf tissue, the half-life was approximately 3 hours and 95% of labeled ABA wa s metabolized after 24 hours (Loveys, 1984). The fastest metabolism of exogenously applied ABA was reported using feeding studies to petioles of cherry leaves ( Prunus sp.), with a half-life of 36.2 5.1 minutes (Gowing et al., 1993). One hypothesis for the faster rate of metabolis m reported when ABA was fed to the petiole versus applied to leaf tissue is the location of ABA relative to the degradative enzymes. In the

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22 absence of stress, leaf-applied ABA is sequestered in the chloroplasts due to high stromal pH while xylem-derived ABA arrives in the apoplas tic space and moves into the cytoplasm, the cellular location of the degrada tive enzymes. Studies observing ABA metabolism in response to water stress cycles have reported a lag time of one and a half hours for ABA levels to start decreasing (Zeevaart 1980). The rec overy period required for ABA leve ls to return to pre-stress levels vary from hours to days: three to four hours for Xanthium (Zeevaart, 1980); one day for grapevine ( Vitis vinifera ) (Loveys and Kriedemann, 1973) and sorghum ( Sorghum bicolor ) (Beardsell and Cohen, 1975); and two days for Brussels Sprouts (Brassica oleracea ) (Wright, 1978), maize ( Zea mays ) (Beardsell and C ohen, 1975), and pea ( Pisum sativum) (Dorffling et al., 1974). Movement To understand how abscisic acid functions as a plant horm one, it is important to understand how it moves from the site of synthesis to the site of action. Abscisic acid can be synthesized in both leaf and root tissue and ha s been shown to accumulate in all plant parts (Shindy et al., 1973). Long distance movement occurs in both xylem and phloem while short distance movement occurs in the apop lastic space and the cytoplasm. Early investigations studying ABA applied exogenously either by sp ray or stem/petiole introduction reported differential plant res ponse between application methods. It was hypothesized that the differences were due to lack of sufficient pene tration of leaf tissue, rate of movement through the tissue, or rapid compartm entalization or metabolism during transport. ABA is capable of penetrating the leaf cuticle and remains biologically ac tive, but the rate is slowed relative to the thickness of the wa xy layers (Blumenfeld and Bukovac, 1971, 1972). Differences in response were most likely attributed to a combination of factors including slower

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23 movement across the cuticle, am ount of ABA compartmentalized in the leaf tissue, and amount of ABA actually reaching the active site. Movement of ABA in plant tissue was ini tially investigated by Dorffling and Bottger (1968) who reported a rate of 24 to 36 mm/hr in Coleus petiole segments. Ingersoll and Smith confirmed this result in cotton ( Gossypium hirsutum ) and reported a rate of 20 to 30 mm/hr in their initial study (1970) and a re vised rate of 22.4 mm/hr, whic h was independent of petiole length or applied ABA concentration, in a second study (1971). In contrast, rate of movement in vascular tissue can be 7 to 10 cm/hr. AB A moves through the plan t by vascular and nonvascular pathways, and actual rate of ABA movement through the plant depends on the pathway or combination of pathways used. Signaling Sequence Abscisic acid is produced in both leaf and root tissue in response to water stress through a com bination of hydraulic and chemical signals. In leaf tissue, the initial signal has been identified as zero turgor pressure (Pierce a nd Raschke, 1980). In root s, drying soil surrounding the root tips is sensed and ABA is synthesized which is translocated to the shoots (Tardieu and Davies, 1993). During stress situations, re-distribution of foliar ABA, up-regulation of synthesis in leaves and roots, and/or translocation from the roots significantly increase ABA concentration by 30-fold surrounding the guard cells (Pei and Kuchitsu, 2005). Under normal water status, 80 to 90% of leaf ABA is contai ned in mesophyll chloroplasts (Loveys, 1977). Synthesis occurs in the cytoplasm of the leaf mesophyll (Hartung, et al., 1981) and distribution is determined by pH gradients. ABA as an un-di sassociated molecule is capable of crossing membranes and moves into the chlopopl ast, where it disassociates to ABAand is trapped due to high stromal pH. Under water stress, stromal pH decreases and releases ABA (Hartung, 1980, 1981; Cowan, 1982), although the signaling mechanism is not known. ABA

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24 accumulates in the apoplast, even after leaves begin synthesis (Cornis h, 1985), and is available for use by the guard cells. The site of action for ABA is the outer pl asmalemma of the guard cells (Hartung, 1983). The plasma membrane is impermeable to ABA in its anion form (ABA-) but ABA-binding proteins in the membrane have their active si tes facing towards the apoplastic space (Hornberg, 1984), enabling them to respond quickly to changes in apoplastic ABA levels. Binding of ABA to the proteins results in an efflux of up to 85% of K+ from the guard ce lls (Outlaw, 1983), resulting in decreased turgor a nd stomatal closure. Some sec ondary messengers that may also play a role in stomatal closure are calcium and pH (Pei and Kuchitsu, 2005). Reports have indicated lag pe riods exist both between initia l stomatal closure and an increase in ABA levels and between removal of water stress and stomatal re-opening. Redistribution of ABA from the chloro plast to the apoplast allows ABA to be present at the site of action before any synthesis occurs. If the water stress continues over a period of time, synthesis occurs to replenish available ABA supply. Afte r water stress has been lifted, continued uptake of ABA occurs from the apoplastic pool even after synthesis has stopped (Raschke, 1975; Ackerson, 1980) and continues until ABA has b een sequestered in the chloroplasts or metabolized. Stomata will not re-open until the AB A concentration has fallen to pre-stress levels (Cornish, 1985). Role in Stomatal Closure Abscisic acid has been shown to play an im portant role in regulating plant water status under stress conditions b y controlling stomatal aper ture and plant transpiration rate. The first report was by Little and Eidt ( 1968) who were investigating the effects of ABA on dormancy of coniferous and deciduous trees and observed a simultaneous inhibition of bud break and transpiration. Mittelheuser and Van Steveninck (1969) reporte d application of exogenous (RS)-

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25 ABA inhibited transpiration in wheat and observed stomatal closure in wheat and barley. They also showed stomatal response to ABA was rapi d and reversible, with closure within ten to fifteen minutes of application and re-opening as the ABA soluti on was diluted (1971). Results from Jones and Mansfield (1970) indicated that st omatal closure was due to effects on guard cell functioning and not due to CO2 concentration. Further studies investigating stomatal functioning using exogenously applied ABA are listed in Table 1-1. Conclusive evidence of the re lationship between abscisic ac id and stomatal functioning was obtained using the ABA-deficient tomato mutant flacca Compared to the normal phenotype of Rhinelands Ruhm, flacca contained a ten-fold lower c oncentration of ABA and wilted rapidly because of abnormal stomatal functioning (Tal and Imber, 1970). Applications of ABA to flacca resulted in normal stomatal functioning a nd normal phenotypic appearance, but plants reverted to the wilty phenotype within a few da ys after applications ceased (Imber and Tal, 1970). In addition to studies inves tigating the effects of abscisic acid applied exogenously, research has also demonstrated that plants produce and/or accu mulate endogenous abscisic acid in response to water stress. Wright and Hiron (1969) observed in excised wheat leaves a fortyfold increase in leaf ABA content after a few hours of wilting. In Brussels sprouts ( Brassica oleracea ), Wright and Hiron (1972) obser ved an increase in the ABA le vel and leaf resistance as the plants wilted. After plants were re-watere d, stomata returned to normal functioning as the ABA level decreased over a few da ys to the pre-stress level. Abscisic Acid as a Chemical Signal Initial ob servations indicated that ABA accumulation in leaf tissue rose as the turgor pressure of leaf cells reached zero (Pierce a nd Raschke, 1980). Many researchers noted that the rapid and significant increase in le af ABA content occurred faster than it could be synthesized.

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26 It was hypothesized that ABA coul d be 1) released from storag e in the chloroplasts or 2) translocated from other parts of the plant, such as the roots. Both theories are correct, but serve different functions. In response to rapid leaf dehydration, change s in stromal pH releases ABA from the chloroplasts, making it available for the guard cells (H artung et al., 1980). Translocation of ABA from root tissue acts as a signal of impe nding water deficit and acts to close stomata before any significant change in le af water status occurs to optimize water use (Zeevaart and Creelman, 1988). Abscisic acid had been hypothesized to be th e chemical signal transl ocated from the roots to the shoots under decreasing water availability due to the re lationship between xylem [ABA], stomatal conductance, and transp iration rate. Many studies atte mpted to demonstrate causation and not merely correlation between xylem [ABA] levels and observed plant response. One of the first requirements was to show that a plant has the capacity to differentiate between leafand root-derived ABA (Davies and Zhang, 1991). When leaf tissue is not st ressed, leaf-derived ABA is usually sequestered in the ch loroplasts due to a pH gradient Root-derived ABA arriving via the transpiration stream is unloaded at the site of evaporation, the cell walls adjacent to guard cells (Meidner, 1975), and close to the site of action, which are the guard cells (Hartung, 1983). It is also important to dem onstrate that enough ABA moves in the transpiration stream to account for the observed plant response (Davies et al., 1994). ABA moves in the xylem with the transpiration stream and preventing transpiration by covering leaves with tinfoil resulted in no increase in ABA levels in leaf tissue (Zhang and Davies, 1987). Feed ing studies in maize (Zhang and Davies, 1990) reported that increased xylem [ABA] correlated with decreased leaf conductance. Similar results occurred when AB A was injected into the stem (Tardieu and

PAGE 27

27 Davies, 1993). When ABA was removed from colle cted xylem sap and fed back to plants, all anti-transpirant activity disappear ed (Zhang and Davies, 1991). The final step in proving the hypothesis wa s to demonstrate causation between xylem [ABA] and reduced stomatal conductance, whic h was reported using tw o split-root system experiments. Zhang and Davies (1989) kept one column well watered and one allowed to air dry. Stomatal conductance for partially-watered plants was reduced after six days, but leaf turgor was never less than the well watered plants. A split-root system using apple (Gowing, et al., 1990) finally demonstrated the role of a positive signal from the roots controlling leaf conductance. Roots were set up in three split -root treatments: well-watered, partially-watered with re-watering after 24 days, and partially-wa tered with drying roots removed after 24 days. During the drying cycle, leaf turgor of partially-watered plants was never lower than the wellwatered plants. After re-watering and root re moval, both groups of plants recovered and responded similarly to well-watered plants. Re-w atering restored the wate r status of the roots and removed the need to produce a root-sourced signal and root excision removed the site of synthesis. Agricultural Importance W ith the discovery of abscisic acid as a plan t hormone responsible for controlling stomatal function relative to plant water relations, there was much discussion of ABA as the ideal antitranspirant for agriculture use. An ideal agent sh ould be: (a) non-toxic for use on ornamentals and food crops, (b) not permanently damage the stomatal mechanism, (c) act specifically and selectively on guard cells, and (d) persist for an extended period of time (Jones and Mansfield, 1972). ABA meets most of the listed criteria but there has been difficulty in obtaining a commercially available supply due to structural inactivation in light, in activity of the (R)

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28 enantiomer, a high cost of production, and difficulty obtaining sufficient quantities naturally or synthetically (Addicott and Lyon, 1969; Milborrow, 1969; Gianfagna, 1995). Work with ABA analogs has been promising as anti-transpirants on marigold ( Tagetes petula ) and tomato (Lycopersicon esculentum ) (Sharma et al., 2005, 2006). Comparison with (RS)-ABA demonstrated that both the analogs and synthetic ABA were effective in reducing plant water usage. However, when compared to the analogs, (RS)-ABA had a shorter efficacy period (Sharma et al., 2006) and negative effects on plant aesthetics, including superficial wilting of tomato under normal water status (Sharm a et al., 2006), flower drop on impatiens ( Impatiens walleriana ) (Gibson and Crowley, 2006) and bedding pl ants (Blanchard et al., 2007), and yellowing of older, lower leaves (Barrett and Campbell, 2006). A formulation of (S)-ABA is currently in tes ting by Valent BioSciences for use as an antitranspirant and holding agent for flor iculture crops. Initial studies show that (S)-ABA applied to bedding plants reduced transpiration and increased postharvest longevity (Stamps and Chandler, 2005; Barrett and Campbell, 2006; Gibson a nd Crowley, 2006; Blanchard et al., 2007).

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29Table 1-1. Summary of studies applying exogenous ABA and evaluating effects on stomatal closure Source Crop Application method ABA concentration Lag Time Time to complete closure Time to recovery Jones and Mansfield (1970) Xanthium pennsylvanicum Leaf surface 10-4 M (0.02 g/cm2) 2-9 days Cummins et al. (1971) Barley ( Hordeum vulgare) Petiole 10-7 M 5 min 5 min after removal Loveys (1984) Grape ( Vitis vinifera ) 2 to 8 x 10-11 M/cm2 30 min 4 to 5 h Horton (1971) Vicia faba Isolated epidermal strips 1.9 x 10-5 M (+)-ABA (10 mg/L) Kriedemann et al. (1972) Bean (Phaseolus vulgaris ) Petiole 8 min 30 min Broadleaf Dock ( Rumex obtusifolia ) Petiole 8 min 35 min Beet ( Beta vulgaris ) Petiole 9 min 52 min Corn (Zea mays ) Petiole 3 min 105 min Rosa spp. Petiole 32 min 108 min Itai et al. (1978) Fed to trans. stream Petiole 9 min

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30 CHAPTER 2 WHOLE PLANT RESPONSE OF CHRYSANTHEMUM TO PACLOBUTRAZOL AND CHLORME QUAT CHLORIDE AS A FUNCTION OF EXPOSURE TIME USING A SPLITROOT SYSTEM Introduction Plant growth regulators (PGRs) which inhi bit production of gibbe rellins and inhibit internode elongation are im portant in the producti on of many floriculture crops. Paclobutrazol and chlormequat chloride are two of these chemical s and are often applied either as a foliar spray or as a substrate drench. Media applications have become increasingly popular due to ease of application, uniformity of effect, and duration of efficacy. However, little is understood of the timing of movement into the pl ant following drench applications to peat-based media commonly used in container production. Pacl obutrazol has relatively low so lubility in water and is only xylem mobile. It is known that paclobutrazol binds to organic media components following drench applications. Chlormequat chloride is highly water soluble and moves in both the xylem and phloem. The split-root system used in this study wa s developed to evaluate chemical uptake in a practical and efficient whole plant system, simila r to commercial application situations. The objective of this study was to evaluate uptake of paclobutrazol and chlormequat chloride applied as a media drench and determine when uptake occurred. Materials and Methods Bare-root cuttings of Dendranthema x grandiflora Snowm ass were obtained from commercial sources (Yoder Brothers, Barberton, Ohio ). Roots of each cutting were cleaned and loosely separated into two equal parts. Cuttings were planted in a 2-cell pack with each half of the roots in separate, adjoining cells. Pl anting medium was Fafard 2 (Apopka, Florida) consisting of 65 peat: 20 perlite: 15 vermiculite. Newly planted cuttings were placed in a mist

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31 house for one week to establish and then were m oved to a fan and pad greenhouse. Plants were pinched to three nodes two weeks after planting and all lateral s hoots except the t op lateral were removed two weeks after pinch. Plants were grown under a non-inductive photoperiod with night interruption lighting from 2200 HR to 0400 HR. Plants were fertilized at each irrigation with a 20N-4.4P-16.6K (Peters fertilizer) with N at 300 mgL-1. Treatments began once plants were approximately 12 cm tall. The chemical being studied was applied to one half of the root system in 20 mL of solution. The treated half of the root system was then excised at prescr ibed time intervals to terminate the plants exposure to the chemical. At each prescribed excision time another set of control plants, which had been drenched with onl y water, was included. Roots of plants for the longest time interval were not excised. Following root excision of the last treatment, plants were planted into 11.5 cm pots using Fafard 2 medium and returned to the greenhouse for the duration of the experiment. This allowed all plants to have the same soil volume for the duration of the experiment and to maintain adequate wate r availability as the plants grew. Chemical uptake during the exposure interval was determined as a function of plant response. Plant height (cm) was recorded on th e day of application and the last day of the experiment. Stem elongation was calculated as the difference between initial and final plant height. To standardize for possible growth reduction caused by root removal, stem elongation of each treated plant during an exposure interval was compared to the corresponding control block average. Results are given as stem elongation as a percent of the control. Data were analyzed using ANOVA and Tukeys mean separation. Re gression analysis was conducted as a NLIN procedure to calculate a li near-plateau model (SAS 9.1, Cary, North Carolina).

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32 Paclobutrazol was applied at a concentration of 6 mgL-1 so each plant received 0.12 mg active ingredient (a.i.). Plants exposed to paclobutrazol were separated into six treatments consisting of exposure times of 1, 2, 4, 8, 12, or 24 days. Exp. 1 was initiated on 9 Sept. and repeated on 4 Nov. 2006. Results from each trial we re very similar and data was pooled. Plants were set up in a randomized complete block design, with three blocks an d three replicates per treatment. Chlormequat chloride was applie d at a concentration of 15,000 mgL-1 for Exp. 2, with each plant receiving 300 mg a.i., and 20,000 mgL-1 for Exp. 3, with each plant receiving 400 mg a.i.. More chemical was used to compensate for faster growth under higher temperatures. Exp. 2, initiated on 31 May 2007, had 6 treatments cons isting of exposure times of 1, 2, 4, 6, 8, or 16 days. Plants were set up in a randomized comp lete block design, with four blocks and three replicates per treatment. Because exposure of one day or longer showed maximum response in Exp. 2 Exp. 3, initiated on 20 July, had 7 treatm ents consisting of expos ure times of 15 min, 30 min, 1 h, 2 h, 4 h, 1 day, and 16 days. Plants we re set up in a randomized complete block design, with three blocks and three replicates per treatment. Results and Discussion The split-ro ot system was an effective model to evaluate chemical uptake on a whole plant level. Chrysanthemum was an effective test plant because plants grew rapidly, remained vegetative under photoperiod manipulation, were responsive to paclobutrazol and chlormequat chloride, and had a uniform response across each treatment. Plants were not significantly affected by root excision (N.S. at 0.05), with the 1 and 24 day controls from the second paclobutrazol experiment having stem elonga tion of 20.1 cm and 21.5 cm, respectively, a difference of 1.4 cm (Table 2-1). This small di fference could be explai ned by the difference in root system size and availabl e water for maximum growth.

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33 Paclobutrazol was taken up slowly from the media, with longer e xposure resulting in greater inhibition of stem elongati on (Figure 2-1.). Data for th e two experiments were similar and pooled together for analysis. Data were regr essed and fit to a linear-p lateau model with y = % stem elongation and x = paclobutrazol exposure (days). The amount of stem elongation was reduced as exposure time following the drench ap plication increased and reached a plateau at 15.85 days (.90) with elongation 44% of the control. In contrast, chlormequat chloride was take n up rapidly from the media over a relatively short period. Plants were exposed to chlormequa t chloride for 1 to 16 days in Exp. 2 and stem elongation was reduced by all chlormequat chloride treatments (Table 2-2). However, there was little difference in stem elongation (between 51 and 55% of the control and N.S. at 0.05) for the different exposure intervals, indicating that amount of chlormequat chloride needed for maximum efficacy was taken up by the plants within 24 hr of application. Exp. 2 was conducted to evaluate the importance of uptake during s horter exposure times (Figure 2-2). Data was regressed and fit to a linear-p lateau model with y = % stem elongation and x = chlormequat chloride exposure (hours) Uptake was rapid and reached a plateau at 6.78 hours (.91) with elongation 55% of the control. The results of Exp. 3 agree with observations in Exp. 2 that chlormequat chloride taken up during the first day of exposure had greater efficacy than chemical taken up between days 1 and 16. These studies show a difference in the patter n of uptake following dren ch applications of paclobutrazol and chlormequat chloride. Paclobut razol is xylem active a nd a strong inhibitor of stem elongation and commonly used as a spray, drenc h, or liner dip for growth control. Results from this study indicating slow uptake explain some of the findings of others and grower observations. This study has demonstrated th at environmental conditions at the time of

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34 application, such as temperature, humidity, a nd light level probably do not influence chemical uptake and efficacy. Efficacy of media appl ied paclobutrazol depends on media composition, amount of chemical (a.i.) applied, irrigation me thod, and species and/or cultivar sensitivity. Paclobutrazol has a low water solubility of 35 ppm (Lever, 1986) and there is a hydrophobic attraction between non-polar portions of th e growth retardant a nd waxes of the bark particles (Barrett, 1982). Paclobut razol is partitioned out of solu tion to the waxy layers of the bark, but can still be obtained, absorbed, and ut ilized by the plant. Th ese binding reactions are thought to occur very rapidly because no loss of efficacy was observed due to leaching during application (Million et al., 1999) or irrigation as soon as one hour af ter application (Barrett et al., 1987). Adsorption reactions generall y take place in the upper levels of the soil and media, above the root zone (Barrett, 1982). Over time, ther e was a slow re-distribution to the middle and lower layers of the media (Million et al., 1999). Paclobutrazol is not ra pidly broken down and has a relatively long half-life, resulting in re sidue issues in certain commercial situations. Efficacy is reduced in media containing pine bark and influenced by type and particle size. Old composted bark, fresh pine bark, and compos ted pine bark required a 4-, 5-, and 10-fold higher concentration of paclobutrazol to achieve similar he ight reduction as Sphagnum peator coir-based media. Finer bark particles redu ce efficacy more than medium or coarse bark particles, presumably due to th e larger surface area av ailable for adsorption (Million et al., 1998). In contrast to paclobutrazol, chlormequat chlo ride is a weaker inhibitor of stem elongation when applied as a media drench, requiring 400 mg a.i. vs. 0.12 mg a.i. Chlormequat chloride is highly water soluble (Cathey and Stuart, 1961) and xylem and phloem mobile. It can be applied as a spray or drench, with drenches having gr eater uptake and efficacy (Belzile and Vonk, 1972).

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35 Chlormequat chloride is rapidly absorbed by pl ants in experiments when it is added to the nutrient solution. In one study, wheat plants were exposed to nutrient solution plus chlormequat chloride for six hours and then returned to normal nutrient so lution. After two weeks, 20% growth inhibition was reported (Dekhuijzen and Vonk, 1974). Becau se of its complete water solubility (MSDS label, 2008), chlormequat chlori de should remain in solution and not readily partition to media components, a llowing it to be taken up by mass flow with the transpiration stream. Efficacy of chlormequat chloride is dependent on amount of active ingredient applied and species and/or cultivar sensitivity. The chemical has a high affinity for remaining in solution and should remain available for uptake as long as it is not washed out of the medium. Based on the results of this study, chlormequat chloride has th e potential to be infl uenced by environmental conditions at time of application. Factors that could influence transp iration, such as light, temperature, and humidity, could affect the rate of uptake as the chemical moves into the plant by mass flow. These factors were not evaluated a nd could be a potential for future research. In summary, plant response to paclobutrazol indicates slow uptake, which is due to rapid partitioning to media components and slow desorp tion in an active state until the chemical pool is sufficiently depleted. Efficacy is dependent on the amount of active ingredient reaching the root zone of the plant an d related to the percent, type, and pa rticle size of bark in the medium. Response to chlormequat chloride indicates ra pid uptake due to the chemical remaining in solution and not interacting with media compone nts. Uptake occurs by mass flow due to transpiration and could be influenced by environmental conditions at time of application that regulate plant water status, including light, temperature, and humidity.

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36 0 0.2 0.4 0.6 0.8 1 0481 21 62 02 4Time (day)Elongation (% of control)y = 0.96 0.033x ; for all points x < x0x0 = 15.85 Figure 2-1. Chrysanthemum elongation response to exposure time following paclobutrazol media drench to one half of root system. Exposure time was time from application to excision of treated roots. Elongation is expressed as percent of control plants with roots excised at same time. The best-fit regression was a linear plateau model y = 0.96 0.033x and the join point at 15.85 days (n = 9).

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37 0 0.2 0.4 0.6 0.8 1 0481 21 62 02 4Time (hour)Elongation (% of control)y = 0.75 0.029x ; for all points x < x0x0 = 6.78 Figure 2-2. Chrysanthemum elongation response to exposure time following chlormequat ch loride media drench to one half of root system. Exposure time was time from app lication to excision of tr eated roots. Elongation is expressed as percent of control plants with roots ex cised at same time. The best-fit regre ssion was a linear plateau model y = 0.75 0.0229x and the join point at 6.78 hours (n = 9).

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38 Table 2-1. Plant elongation response following application of water to one half of root system. Treatment was exposure time from applicati on to excision of r oots. Data were analyzed using ANOVA and di fferences were N.S. at 0.05 (n = 9). Treatment Control elongation (cm) 1 day 20.1 2 days 20.7 4 days 22.6 8 days 21.8 12 days 21.6 24 days 21.5 Table 2-2. Plant elongation response to exposur e time following chlormequat chloride media drench to one half of root system. Exposure time was time from application to excision of treated roots. Elongation is expr essed as percent of control plants with roots excised at same time. Data were analyzed using ANOVA and determined to be N.S. at 0.05 (n = 12) (Exp. 2) Treatment Elongation (% of control) 1 day 51 2 days 52 4 days 55 6 days 51 8 days 51 16 days 53

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39 CHAPTER 3 WHOLE PLANT RESPONSE OF CHRYSANTHEMUM TO (S)-ABSCISIC ACID AS A FUNC TION OF EXPOSURE TIME USING A SPLIT-ROOT SYSTEM Introduction Abscisic acid (ABA) is responsible for controlling stom atal function. Under water stress, increased concentration of ABA accumulates at the guard cell, due to re-distribution from chloroplasts, increased synthesis by leaf tissue, or transport from root tissue (Pei, 2005). Exogenous applications of ABA to detached leav es and whole plants with normal water status have resulted in stomatal closure and decreased transpiration (Mittelheuser and Van Steveninck, 1969). It has been proposed that ABA would be an ideal anti-transpiran t for agriculture use under field conditions to reduce water usage and irrigation requirements (Jones and Mansfield, 1972), but this has not occurred due to the hi gh cost of synthesis, production of a racemic mixture, and difficulty obtaining sufficien t quantities (Addicott and Lyon, 1969; Gianfagna, 1995). (RS)-ABA is effective in controlling ot her plant physiological processes, but stomata respond only to (S)-ABA (Kriedemann et al., 1972). Development of commercially available (S)-ABA has created interest among the horticulture industry as a holding ag ent to increase postharvest longevity of ornamentals. Many high-value potted floriculture cr ops are subjected to water stress during shipping and display at retail, decreasing their quality and va lue. Recent studies on hibiscus ( Hibiscus rosa-sinensis), impatiens (Impatiens walleriana), and bedding plants indicate that treatment with (S)-ABA increases postharvest longevity. Because of the nature of the hormone, care must be taken when applying to avoid flower drop or yellowing of older, lower leav es (Stamps and Chandler, 2005; Barrett and Campbell, 2006; Gibson and Crowley, 2006; Blanchard et al., 2007).

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40 Most studies evaluating ABA uptake have used labeled material and results were obtained from radiography or isolation and extraction procedures. The split-root system used in this study was developed to evaluate ABA uptake in a practi cal and efficient whole plant system, similar to commercial application situations. The objective of this study was to evaluate the uptake of ABA applied as a media drench and determine how fast uptake from the media occurs. Materials and Methods Bare-root cuttings of Dendranthema x grandiflora Snowm ass were obtained from commercial sources (Yoder Brothe rs, Barberton, Ohio) and grown according to procedures in Ch. 2. Plans were not exposed to water stress during the growth period and treatments began when plants were approximately 15 cm tall. (S)-abs cisic acid was applied to one half of the root system at a concentration of 2,000 mgL-1 (determined during an initia l rate screen) in a volume of 20 mL so each plant received 40 mg of active ingredient (a.i.). The treated half of the root system was excised at different time intervals to stop the plants exposure to the chemical. Plants exposed to (S)-ABA were separated into 7 treatments consisting of exposure times of 7.5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and an uncut plant. Plants in two control treatments of water only were included, one with half the root system removed and one with a complete root system. Abscisic acid was applied at 1100 HR to well-watered plants in a high light environment and water was withheld for the duration of the experiment. ABA efficacy was determined by measuring transpiration rate s using a LI-1600 steady-state porometer (LI-COR, Lincoln, Nebraska). The first experiment was conducted on 7 Se pt. 2007 and data were collected four hours after (S)-ABA application. Light level at time of transpiratio n measurement was 950 mol m-2s-1. The second experiment was initiated on 5 Oct. 2007 and data were collected 24, 48, and

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41 72 hr after (S)-ABA application and light levels at time of measurement were 250, 600, and 700 molm-2s-1, respectively. The third experiment wa s initiated on 2 Jan. 2008 and data were collected at 4, 24, and 48 hours after (S)-ABA a pplication and light levels at time of measurement were 600, 700, and 600 molm-2s-1, respectively. Experiments were set up in a randomized complete block design, with two blocks and six plants per treatment. Data were analyzed using GLM and Waller-Duncan for mean separation (SAS 9.1, Cary, North Carolina). Results and Discussion Transpiration rates fo r control plants without and w ith root excision were 15.4 and 10.9 g cm-2s-1, respectively, in Exp. 1 (Figure 3-1). Ther e was a difference in tr anspiration that can be attributed to root excision and could possibly be due to a decrease in available water or a direct response to the root ex cision. However, transpiration of plants with excised roots was considerably greater than transpiration of plants given (S)ABA. In Exp. 2 and 3 there was a relatively smaller difference in transpiration for th e plants in the control treatments (Figures 3-2 and 3-3). For the first experiment (Figure 3-1), data were collected 4 h afte r application of (S)abscisic acid. All plants treate d with (S)-ABA had decreased tran spiration rates compared to the controls. (S)-ABA uncut plants had leaf transp iration of 0.89 gcm-2s-1and plants in (S)-ABA treatments with exposure intervals betw een 7.5 min and 4 h were between 0.7 and 2.68 gcm2s-1. Although the exposure interval of 7.5 min redu ced transpiration compared to the control, transpiration in these plants was greater than in the other ABA treatments. (S)-ABA was taken up by the roots from the medium and moved into the xylem or stem tissue very rapidly over a period of minutes. Results from this experiment indicate that the plan ts took up sufficient (S)ABA in a short exposure period to close stomata within 4 h of application, making this an

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42 effective model system to test (S)-ABA uptake. However, it was unknown if the amount of (S)ABA taken up during shorter exposur e intervals was sufficient to provide the same duration of stomatal closure compared to longer exposure intervals. Exp. 2 and 3 were conducted to answer th e above question. Transpiration for Exp. 2 recorded at 24, 48, and 72 h after (S)-ABA appl ication. The time from ABA application to excision of roots did not affect tr anspiration rate measured at 24 h, but there were differences at 48 and 72 h (Fig. 3-2). At 24 h, lower transpir ation rates for all (S)-A BA exposure intervals were observed, compared to both co ntrols, and were between 1.14 and 1.72 gcm-2s-1. At 48 h after application, transpirati on rates for exposure intervals of 7.5 and 15 min were 5.35 and 6.56 gcm-2s-1, respectively, which were greater than transpira tion rates between 2.09 and 3.26 gcm-2s-1 for all intervals between 30 min and 4 h. At 72 h after application, transpiration rates for all (S)-ABA exposure intervals were reduced co mpared to the uncut cont rol. The cut control was not included because plants were wilted. Although all cut (S)-ABA treatments were not significantly different from the unc ut (S)-ABA treatment, there wa s a general trend of decreasing transpiration as exposure interval increased. Data from Exp. 3 confirms findings from Exp. 1 and 2 (Fig. 3-3). At 4 h after application, transpiration rates of all (S)-ABA exposure intervals were less th an for water controls. The only difference between experiments was the reduced efficacy of the 7.5 min exposure interval compared to all other (S)-ABA exposure interv als in Exp. 1. At 24 h after application, transpiration rate of the 7 .5 min exposure interval was 4.43 gcm-2s-1, which was greater than transpiration rates between 1.97 and 2.51 gcm-2s-1 for exposure interv als between 30 min and 4 h. At 48 h after application, transpiration rates for exposure intervals of 7.5 min, 30 min, and 2

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43 h were 5.49, 4.18, and 3.53 gcm-2s-1, respectively, indicating that longer exposure intervals were needed to achieve maximum stomatal closure over the duration of the effect. Efficacy of a chemical is dependent on the amount present at the active site. The concentration of ABA surrounding th e guard cells is in continual flux because of movement from the roots, distribution between cellular compartm ents, synthesis of new ABA, and metabolism of existing ABA (Pei and Kuchitsu, 2005). In th e experimental system, our simplified model assumes: 1) only xylem transport of (S)-ABA absorbed from the medium, 2) metabolism of endogenous (RS)and exogenous (S)-ABA during the duration of the experiment, and 3)ignores synthesis and re-distribution because plants ar e not subjected to water stress or pressure dehydration. Because uptake is stopped with root excision, a fixed amount of (S)-ABA is translocated to the guard cells and ava ilable to regulate st omatal function. Experimental results indicate that sufficient (S)-ABA was applied to close stomata and decrease leaf transpiration ra te. (S)-ABA was taken up by the roots from the media and moved into the xylem or stem tissue ve ry rapidly over a period of minut es. This experiment did not measure rate of movement through the plant and/ or stomata reaction time, but 4 h was sufficient for (S)-ABA to move through the plant to the guard cells and cause stomatal closure. It is unknown how rapid ABA movement is into the plant from soil ap plication. However, early studies observed rates of stomatal closure by applying exogenous ABA to detached leaves by placing the petiole in a solution c ontaining ABA. Complete stomatal closure was fastest for bean ( Phaseolus vulgaris ), requiring 30 min. and slowest for rose ( Rosa spp.), requiring 108 min. (Kriedemann, 1972). Other studies looking at movement of ABA through petiole segments reported rates of 24 to 36 mm/hr in Coleus (Dorffling and Bottger, 1968) and 22.4 mm/hr in

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44 cotton ( Gossypium hirsutum ) (Ingersoll and Smith, 1971), but the rate of movement in vascular tissue can be 7 to 10 cm/hr. Increased leaf transpiration rates of all (S)ABA exposure intervals over the efficacy period (Figs. 3-2 and 3-3) can possibly be explained by metabolism. Studies observing rate of metabolism have reported that 32% and 64% of leaf applied 14C-ABA was metabolized after 8 and 24 hours, respectively (Hartung et al ., 1980). The half-life in grape ( Vitis vinifera ) leaftissue was approximately 3 hours and 95% of labeled ABA was metabol ized after 24 hours (Loveys, 1984). Recent studies investigating media applied AB A reported that water use was suppressed for three days in tomato plants ( Lycopersicon esculentum ) (Sharma et al., 2006). Also, the trend of increasing leaf transpiration as exposure interval decreases can be explained as a smaller concentration of (S)-ABA taken up and available to elicit maximum stomatal response. This research indicates that (S)-ABA applied as a drench to peat-based media is taken up very rapidly and causes stomatal cl osure within 4 h. This is in contrast to results obtained for paclobutrazol where uptake was slow, with longer exposure resulting in greater inhibition of stem elongation (Ch. 2). Regressi on analysis was significant as a linear plateau model, reaching a plateau at 15.85 days (.90) with elongation 44 % of the control. Media composition, amount of chemical applied, and species/cultivar sens itivity can influence paclobutrazol efficacy. Even though (S)-ABA is taken up rapidly, expos ure intervals of one hour or greater are needed to achieve maximum reduction in leaf tr anspiration over the efficacy period of 48-72 h. Stomatal response is dependent on the amount of ABA present at the guard cells (Kriedemann et al., 1972). The minimum exposure interval needed to achieve maximum stomatal response could differ depending on factors influencing uptake. Possible factors to investigate are the concentration of (S)-ABA app lied and the environmental cond itions at application. Our

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45 application concentration was determined by an init ial rate screen, but it is possible that if the concentration was increased, an increase of (S )-ABA could be absorbed in the same time interval, resulting in a shorter e xposure interval needed to achie ve maximum stomatal response at 48-72 h. Also, absorbed (S)-ABA is translocated from roots to shoots in the transpiration stream. If transpiration is redu ced due to light level or other e nvironmental factors at and shortly after application, it is possible that uptake could be reduced, re sulting in a longer time interval needed for maximum stomatal response. Anothe r unknown factor is media moisture level at the time of application, an important parameter for efficacy of liner dips. Plants with reduced moisture level may already be conserving water by stomatal closure and reduced transpiration, potentially slowing the uptake of (S )-ABA with the transpiration stream.

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46 0 2 4 6 8 10 12 14 16 uncutcut7.5 min15 min30 min1 h2 h4 huncutTreatmentTranspiration rate (ugcm-2s-1) Water (S)-AB A a c dcdd cdcd d b Figure 3-1. Effects of (S)-ABA exposure interval on transpiration rate of Dendranthema x grandiflora Snowmass (Exp. 1). Treatment time was time from application to excision of treated roots. Data was recorded at 4 hours after application. Data were analyzed using ANOVA and Waller-Duncan mean separa tion. Letters signify differen ces between treatments at 0.05 (n = 6).

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47 0 2 4 6 8 10 12 14uncutcut7.5 min15 min30 min1 h2 h4 huncutTranspiration rate (ugcm-2s-1)a a bbbbbbb A 0 2 4 6 8 10 12 14uncutcut7.5 min15 min30 min1 h2 h4 huncutTranspiration rate (ugcm-2s-1)bc bc d d d d cd a a B 0 2 4 6 8 10 12 14 uncutcut7.5 min15 min30 min1 h2 h4 huncutTreatmentTranspiration rate (ugcm-2s-1) Water (S)-AB A a bc b cd cd d d cd C Figure 3-2. Effects of (S)-ABA exposure interval on transpiration rate of Dendranthema x grandiflora Snowmass (Exp. 2). Treatment time was time from application to excision of treated roots. Data for cu t control was not recorded at 72 h after application due to plants wilting. Data were analyzed using ANOVA and WallerDuncan mean separation. Letters sign ify differences between treatments at 0.05 and are for comparison within each tim e observation (n = 6). A) 24 h after application, B) 48 h after application, C) 72 h after application.

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48 0 2 4 6 8 10 12 14 uncutcut7.5 min15 min30 min1 h2 h4 huncutTranspiration rate (ugcm-2s-1) b ccccc c c a A 0 2 4 6 8 10 12 14 uncutcut7.5 min15 min30 min1 h2 h4 huncutTranspiration rate (ugcm-2s-1) a c cdddddd b B 0 2 4 6 8 10 12 14 uncutcut7.5 min15 min30 min1 h2 h4 huncutTreatmentTranspiration rate (ugcm-2s-1) Water (S)-AB A a b bcbc cd cdd cdC Figure 3-3. Effects of (S)-ABA exposure interval on transpiration rate of Dendranthema x grandiflora Snowmass (Exp. 3). Treatment time was time from application to excision of treated roots. Data for cu t control was not recorded at 48 h after application due to plants wilting. Data were analyzed using ANOVA and WallerDuncan mean separation. Letters sign ify differences between treatments at 0.05 and are for comparison within each time observa tion (n = 6). A) 4 h after application, B) 24 h after application, C) 48 h after application.

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49 CHAPTER 4 CONCLUSION Plant growth regulators are im portant to the horticulture industry and research has been conducted to determine uptake, translocation, meta bolism, and mode of action. Most research has been conducted using labeled material and re sults determined using radiography or isolation and extraction procedures. Little to no work has been published on the uptake rate of mediaapplied plant growth regulators, an important fa ctor in determining how much chemical exposure time is required to achieve maximum efficacy a nd which portion of the uptake period is most critical for efficacy. Results from this type of study could be used to answer applied research questions concerning the importan ce of environmental conditions at the time of application, differences in plant response relative to type of media application (drenc h vs. sub-irrigation), and the recommendation that drenches be applied in the morning. The objectives of this study were to develop a whole-plant system to evaluate uptake of media-app lied plant growth regulators and to use the system to determine uptake of paclobutrazol, chlorme quat chloride, and (S)-abscisic acid. A split-root system using Dendranthema x grandiflora Snowmass was developed as a model system to determine uptake as a function of plant response. Roots were separated into two compartments of a cell pack. The chemical under evaluation was applied to one-half of the root system, which was excised at prescribed time intervals to terminate the plants exposure to the chemical. Uptake was determined as a functi on of plant response. The system was effective because root excision had little to no effect on the plant. Stem elongation for the 1 and 24 day controls from the second p aclobutrazol experiment was 20.1 cm and 21.5 cm, respectively, a difference of 1.4 cm and statisti cally non-significant. Transpirat ion rates for the non-cut and cut controls from the third (S)-ABA experiment were 9.67 and 8.73 gcm-2s-1 at 4 hours after

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50 application (HAT) and 14.04 and 12.35 gcm-2s-1 at 24 hours after application. Observed differences were due to expos ure interval and theoretically amount of chemical taken up, although this parameter was not determined. Paclobutrazol uptake was determined to be sl ow, with longer exposure resulting in greater inhibition of stem elongation. These results ex plain the findings and observations of other research studies. Paclobutrazo l has low water solubility (Lever 1986) and is not readily leached out of the media by significant amounts of wate r as soon as one hour following application (Barrett et al., 1987). The chemical, as determin ed by bioassay, is generally located in the upper levels of the media, with gra dual re-distribution to lower laye rs over time (Million et al., 1999). There is a hydrophobic attraction be tween the non-polar portions of the molecule and waxes of bark particles (Barrett, 1982). Data from this study propose that the binding reaction occurs rapidly because little inhibition of stem elongatio n was observed from one day of exposure. The reaction is reversible and paclobut razol can still be obtained, abso rbed, and utilized by the plant until the chemical pool has been depleted, whic h follows the observed linear-plateau model. In contrast to paclobutrazol, chlormequat chlo ride uptake was determined to be rapid. Uptake pattern also followed a linear-plateau function and reached a pl ateau at 6.78 hours, in contrast to 15.85 days for paclobutrazol. Chlorm equat chloride is rapidly absorbed by wheat plants when added to nutrient solution, with exposure of six hours result ing in 20% growth inhibition (Dekhuijzen and Vonk, 1974). Chlormequat chloride is highly water soluble (Cathey and Stuart, 1961) and transloca tion of media-applied chemical occurs in the xylem with the transpiration stream. Results from this study sugg est that the chemical re mains in solution, with little interaction with media components, and is readily absorbed by mass flow. Under this assumption, uptake of chlormequat chloride is dependent on transpir ation flux and could

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51 potentially be influenced by factors affecting plan t transpiration, such as light, temperature, and humidity. Uptake of (S)-abscisic acid was also determined to be rapid. Sufficient chemical was absorbed during exposure interv als of 7.5 min to 4 hours to close stomata and significantly reduce transpiration rate at 4 hour s after application. Since (S)-A BA is rapidly metabolized and effects are generally short-lived, experiments were conducted to dete rmine if uptake during shorter exposure intervals was sufficient to pr ovide the same duration of stomatal closure compared to longer exposure intervals. A general trend observed was shorter exposure intervals having reduced efficacy at 24, 48, and 72 hours after application. Differences were more pronounced at the later data observations, with exposure of at least one hour required for maximum efficacy. (S)-ABA efficacy is dependent on the amount present at the active site and, in this model system, is a function of uptake and metabolism. Abscisic acid is rapidly metabolized in leaf tissue (Loveys and Krie demann, 1973; Hartung et al ., 1980,). Uptake could possibly be influenced by application concentr ation and environmental factors that affect transpiration rate. Efficacy of (S)-ABA is co mmercially important and f actors during and shortly after application that in fluence uptake are important to investigate. The split-root system model was used in this study to effectively evaluate the uptake of three plant growth regulators paclobutrazol, ch lormequat chloride, and (S)-ABA. This system answered the initial que stion of how much chemical exposure time was required for maximum efficacy under normal growing conditions. Questions that possible future research could answer is the importance of environmental or other factors during or shortly after application on chemical uptake, especially for chemicals that remain in solution and do not partition to media components. In addition, this system has the pote ntial to be used to answer uptake questions for

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52 other chemical pesticide classes applied as media drenches, incl uding herbicides, fungicides, and insecticides.

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53 WORKS CITED Addicott, F.T., H.R. Car ns, J.W. Cornforth, J.L. Lyon, B.V. Milborrow, K. Ohkuma, G. Ryback, O.E. Smith, W.E. Thiessen, and P.F. Warei ng. 1968. Abscisic acid: A proposal for the redesignation of absc isin II (dormin), p. 1527-1529. In: F. Wightman and G. Setterfield (eds.). Biochemistry and Physiology of Plan t Growth Substances. Runge Press, Ottawa, Canada. Addicott, F.T., J.L. Lyon, K. Ohkuma, W.E. Thiessen, H.R. Carns, O.E. Smith, J.W. Cornforth, B.V. Milborrow, G. Ryback, and P.F. Wa reing. 1968. Abscisic acid: A new name for abscisin II (dormin). Science 159:1493. Addicott, F.T, and J.L. Lyon. 1969. Physiology of abscisic acid and related substances. Annu. Rev. Plant Physiol. 20:139-164. Armitage, A.M. 1994. Ornamental bedding plan ts. CAB International, Wallingford. Arteca, R.N. 1996. Plant growth substances: Prin ciples and applications. Chapman and Hall, New York, N.Y. Barrett, J.E. 1982. Chrysanthemum height cont rol by ancymidol, PP333, and EL-500 dependent on medium composition. HortScience 17(6):896-897. Barrett, J. 2006. PGR trends: New and novel. Greenhouse Product News, Nov.:26-28. Barrett, J.E., and C.A. Bartuska. 1982. PP333 effects dependent on stem elongation dependent on site of application. HortScience 17(5):737-738. Barrett, J.E., C.A. Bartuska, and T.A. Ne ll. 1987. Efficacy of ancymidol, daminozide, flurprimidol, paclobutrazol, and XE-1019 when followed by irrigation. HortScience 22(6):1287-1289. Barrett, J. and C. Campbell. 2006. (S)-ABA: Deve loping a new tool for the big grower. Big Grower, Nov.: 26-29. Beardsell, M.F., and D. Cohen. 1975. Relationships between leaf water status, abscisic-acid levels, and stomatal resistance in maize and sorghum. Plant Physiol. 56(2):207-212. Belzile, L., R. Paquin, and C. Willemot. 1972. Absorption, translocation et mtabolisme du chlorure de (2-chlorothyl) trimthylamm onium-1,2-14C chez lorge dhiver (Hordeum vulgare. Can. J. of Bot. 50:2665-2672. Bier, H., and W. Dedek. 1970. Zur frage des abbaues von 15Nund 14C -chlorcholinchlorid (CCC) in hheren pflanzen. Biochem. Physiol. Pflanzen (BPP) 161:403. Birecka, H. 1967. Transloca tion and distribution of 14C -labelled (2-chloroethyl) trimethylammonium (CCC) in wheat. Bul. de lacademie Polonaise des Sci. 15(11):707714.

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54 Blanchard, M.G., L.A. Newton, E.S. Runkle, D. Woolard, and C.A. Campbell. 2007. Exogenous applications of abscisic acid improved the pos tharvest drought tolerance of several annual bedding plants. Acta Hort. 755:127-133. Blinn, R.C. 1967. Biochemical behavior of 2-chlo roethyl trimethylammonium chloride in wheat and in rats. J. Agr. Food Chem. 15(6):984-987. Blumenfeld, A. and M.J. Bukovac. 1971. Penetration of abscisic acid through enzymatically isolated tomato fruit cuticle. Plant Physiol. 47(S):22. Blumenfeld, A. and M.J. Bukovac. 1972. Cuticular penetration of absc isic acid. Planta 107:261268. Cathey, H.M. 1964. Physiology of growth retarding chemicals. Ann. Rev. Plant Physiol. 15:271302. Cathey, H.M., and N.W. Stuart. 1961. Comparativ e plant growth-retardi ng activity of amo-1618, phosfon, and CCC. Botanical Gazette 123(1):51-57. Cornforth, J.W., B.V. Milborrow, and G. Ryback. 1965. Synthesis of ()-Abscisin II. Nature ( ): 715. Cornforth, J.W., B.V. Milborrow, G. R yback, and P.F. Wareing. 1965. Chemistry and physiology of dormins in sycamore. Nature 205:1269-1270. Cornish, K. and J.A.D. Zeevaart. 1985. Movement of abscisic acid into the apoplast in response to water stress in Xanthium strumarium L. Plant Physiol. 78:623-626. Cowan, I.R. 1982. Regulation of water use in rela tion to carbon gain in higher plants, p. 589-613. In: O.L. Lange, P.S. Nobel, C.B. Osmond, a nd H. Zeigler (eds.). Physiological plant ecology II. Water relations and carbon assim ilation. Encyclopedia of Plant Physiology New Series 12B, Springer-Verlag, London. Cox, D.A. 1991. Gibberellic acid reverses effects of excess paclobut razol on geranium. HortScience 26(1):39-40. Creelman, R.A., and J.A.D. Zeevaart. 1984. Incorporation of oxygen into abscisic acid and phaseic acid from molecular oxygen. Plant Physiol. 75:166-169. Cummins, W.R., H. Kende, and K. Raschke. 1971. Specificity and reversibility of the rapid stomatal response to abscisic acid. Planta 99:347-351. Cummins, W.R., and E. Sondheimer. 1973. Activity of the asymmetric isomers of abscisic acid in a rapid bioassay. Planta 111:365-369. Davies, W.J., F. Tardieu, and C.L. Trejo. 1994. How do chemical signals work in plants that grow in drying soil? Plant Physiol. 104:309-314.

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55 Davies, W.J. and J. Zhang. 1991. Root signals and the regulation of growth and development of plants in drying soil. Ann. Rev. Plan t Physiol. Plant Mol. Biol. 42:55-76. Davis, T.D., and E.A. Curry. 1991. Chemical regulation of vegetative grow th. Critical Rev. Plant Sci. 10(2):151-188. Davis, T.D., G.L. Steffens, and N. Sankhla. 198 8. Triazole plant growth regulators. Hort. Rev. 10:63-105. Dekhuijzen, H.M., and C.R. Vonk. 1974. The distribution and degradation of chlormequat in wheat plants. Pesticide Biochem. Physiol. 4:346-355. Drffling, K., and M. Bttger. 1968. Transport von abscisinsure in explantaten, blattstiel und internodialsegmenten von coleus rh eneltianus. Planta 80:299-308. Drffling, K., B. Sonka, and D. Tietz. 1974. Varia tion and metabolism of abscisic acid in pea seedlings during and after wate r stress. Planta 121(1):57-66. Early, J.D.. 1986. Transport and growth effects of paclobutrazol in p each seedlings, Prunus persica (L.) Batsch. University of California, Davis, MS Thesis. Early, J.D., and G.C. Martin. 1988. Translocatio n and breakdown of 14C-labeled paclobutrazol on Nemaguard peach seedlings HortScience 23(1):196-200. El-Fouly, M.M., and J. Jung. 1969. Some factors affect the degradation of (2-chloroethyl) trimethylammonium chloride by wheat pl ant extracts. Experientia 25:587-588. Gent, M.P.N., and R.J. McAvoy. 2000. Plant growth retardants in ornamental horticulture: A critical appraisal, p. 89-145. In: A.S. Basra (ed.). Plant growth regulators in agriculture and horticulture: Their role and commercial uses. Food Product Press, New York, NY. Gianfagna, T. 1995. Natural and synthetic growth regulators and their use in horticultural and agronomic crops, p 751-773. In: PJ Davies (ed.). Plant hormones: Physiology, biochemistry, and molecular biology. Kluwer Academic Publishers, Dordrecht, Neth. Gibson, J.L., and S. Crowley. 2006. Abscisic acid drenches improve postproduction shelf life of impatiens. HortScience 40(3):511-512. Goulston, G.H., and S.J. Shearing. 1985. Review of the effects of paclobutrazol on ornamental pot plants. Acta Hort. 167:339-348. Gowing, D.J.G., W.J. Davies, and H.G. Jone s. 1990. A positive root-sourced signal as an indicator of soil drying in apple, Malus x domestica Borkh. J. Expt. Bot. 41:1535-1540. Gowing, D.J.G, W.J. Davies, C.L. Trejo, and H. G. Jones. 1993. Xylem-transported chemical signals and the regulation of plant growth and physiology. Ph il. Trans.: Biol. Sci. 341:4147.

PAGE 56

56 Halevy, A.H., and S.H. Wittwer. 1965. Growth promotion in the snapdragon by CCC, a growth retardant. Die Naturwissenschaften 52(11):310 Hartung, W. 1983. The site of action of abscisic acid at the guard cell plasmalemma of Vallerianella locusta Plant, Cell, Environ. 6:427-428. Hartung, W., H. Gimmler, B. Heilmann, and G. Kaiser. 1980. The site of abscisic acid metabolism in mesophyll cells of Spinacia oleracea Plant Sci. Lett. 18:359-364. Hartung, W., H. Gimmler, and B. Heilmann. 1981. Do ch loroplasts play a role in abscisic acid synthesis? Plant Sci. Lett. 22:235-242 Hedden, P. and J.E. Graebe. 1985. Inhibition of gibbe rellin biosynthesis by paclobutrazol in cellfree homogenates of Cucurbita maxima endosperm and Malus pumila embryos. J. Plant Growth Regulat. 4:111-122. Hornberg, C., and E.W. Weiler. 1984. High affinity binding s ites for abscisic acid in the plasmalemma of Vicia faba guard cells. Nature 310:321-324. Horton, R.F.. 1971. Stomatal opening: the role of abscisic acid. Ca n. J. Bot. 49:583-585. Imber, D., and M. Tal. 1970. Phenotypic reversion of flacca, a wilty mutant of tomato, by abscisic acid. Science 169:592-593. Ingersoll, R.B., and O.E. Smith. 1970. Movement of (RS)-abscisic acid in the cotton explant. Plant Physiol. 45:576-578. Ingersoll, R.B., and O.E. Smith. 1971. Transport of abscisic acid. Plant Cell Physiol. 12:301-309. Intrieri, C., and K. Ryugo. 1974. Uptake, transport and metabolism of (2-chloroethyl) trimethylammonium chloride in seedlings of almond ( Prunus amygdalus Batsch.). J. Amer. Soc. Hort. Sci. 99(4): 349-3512. Itai, C., J.D.B. Weyers, J.R. Hillman, H. Me idner, and C. Willmer. 1978. Abscisic acid and guard cells of Commelina communis L. Nature 271:652-653. Jackson, M.J., M.A. Line, and O. Hasan. 1996. Mi crobial degradation of a recalcitrant plant growth regulator paclo butrazol (PP333). Soil Biol. Biochem. 28(9):1265-1267. Jacyna, T., and K.G. Dodds. 1995. Some effects of soil-applied paclobutrazol on performance of Sundrop apricot (Prunus armeniaca L.) trees and on residue in the soil. N.Z. J. Crop Hort. Sci. 23:323-329. Jones, R.J., and T.A. Mansfield. 1970. Suppression of stomatal opening in leaves treated with abscisic acid. J. Expt Bot. 21:714-719. Jones, R.J., and T.A. Mansfield. 1972. Effects of ab scisic acid and its este rs on stomatal aperture and the transpiration ratio. Physiol. Plant. 26:321-327.

PAGE 57

57 Jung, J., and M.M. El-Fouly. 1966. ber den abbau von chlorcholinchlorid (CCC) in der pflanzen. Z. Pfanzenernaehr. Dueng. Bodenk. 114:128. Kriedmann, P.E., B.R. Loveys, G.L. Fuller, a nd A.C. Leopold. 1972. Abscis ic acid and stomatal regulation. Plant Physiol. 49:842-847. Kust, C.A.. 1986. Cycocel plant growth regulan t: Uses in small grains, p. 178-186. In: P MacGregor (ed.). Plant growth regulators in agri culture. Food and Fert. Technol. Ctr for the Asian and Pacific Reg., Taipei, Taiwan. Lever, B.G.. 1986. Cultar A technical overview. Acta Hort. 179:459-466. Li, Y., and Walton, D.C.. 1990. Violaxanthin is an abscisic acid precursor in water-stressed darkgrown bean leaves. Plant Physiol. 92:551-559. Little, C.H.A., and D.C. Eidt. 1968. Effect of abscisic acid on budbreak and transpiration in woody species. Nature 220:498-499. Lord, K.A., and A.W. Wheeler. 1981. Uptake and movement of 14C-chlormequat chloride applied to leaves of barley and wheat. J. Expt. Bot. 32(128):599-603. Loveys, B.R.. 1977. The intracellular location of absc isic acid in stressed and non-stressed leaf tissue. Physiol. Plant. 40:6-10. Loveys, B.R.. 1984. Abscisic acid trans port and metabolism in grapevine ( Vitis vinifera L.) New Phytol. 98:575-582 Loveys, B.R., and P.E. Kriedemann. 1973. Rapid ch anges in abscisic ac id-like inhibitors following alterations in vine leaf water potential. P hysiol. Plant. 28(3):476-479. Meidner, H. 1975. Water supply, evaporation, and vapor diffusion in leaves. J. Expt. Bot. 26:666-673. Milborrow, B.V.. 1969. The occurrence and function of abscisic acid in plants. Sci. Prog. 57:533-558. Milborrow, B.V. 1970. The metabolism of abscisic acid. J. Expt. Bot. 21: 7-29. Milborrow, B.V. 1974. The chemistry and physiology of abscisic acid. Ann. Rev. Plant Physiol. 25:259-307. Million, J.B., J.E. Barrett, T.A. Nell, and D. G. Clark. 1998. Influence of media components on efficacy of paclobutrazol in inhibiting grow th of broccoli and petunia. HortScience 33(5):852-856. Million, J.B., J.E. Barrett, T.A. Nell, and D. G. Clark. 1999. Paclobutrazo l distribution following application to two media as determined by bioassay. HortScience 34(6):1099-1102.

PAGE 58

58 Mittleheuser, C.J., and R.F.M. Van Stevenin ck. 1969. Stomatal clos ure and inhibition of transpiration induced by (RS )-abscisic acid. Nature 221:281-282. Mittleheuser, C.J., and R.F.M. Van Steven inck. 1971. Rapid action of abscisic acid on photosynthesis and stomatal re sistance. Planta 97:83-86. Mooney, R.P., and N.R. Pasarela. 1967. Determina tion of chlorocholine chloride residues in wheat grain, straw, and green wheat foliage. J. Agr. Food Chem. 15:989-995. Ohkuma, K., F.T. Addicott, O.E. Smith, and W.E. Thiessen. 1965. The structure of abscisin II. Tetrahedron Lett. 29:2529-2535. Ohkuma, K., J.L. Lyon, F.T. Addicott, and O.E. Smith. 1963. Abscisin II, an abscissionaccelerating substance from young co tton fruit. Science 142:1592-1593. Outlaw, W.H. 1983. Current concepts on the role of potassium in stomatal movements. Physiol. Plant. 59(2):302-311. Pei, A.M., and K. Kuchitsu. 2005. Early ABA signali ng events in guard cells. J. Plant Growth Reg. 24:296-307. Pierce, M., and K. Raschke. 1980. Correlation be tween loss of turgor and accumulation of abscisic acid in detached leaves. Planta 148:174-182. Rademacher, W. 2000. Growth reta rdants: Effects of gibberell in biosynthesis and other metabolic pathways. Ann. Rev. Plant Physiol. Plant Mol. Biol. 51:501-531. Raschke, R. 1975. Stomatal action. Ann. Rev. Plant Physiol. 26:309-340. Richardson, P.J. and J.D. Quinlan. 1986. Uptake a nd translocation of pacl obutrazol by shoots of M.26 apple rootstock. Pl ant Growth Reg. 4:347-356. Robinson, P.M. and P.F. Wareing. 1964. Chemical nature and biological properties of the inhibitor varying with photoperiod in sycamore (Acer pseudoplatanus). Physiol. Plant. 17:1964. Rothwell, K., and Wain, R.L. 1964. Studies on a growth inhibitor in yellow lupin ( Lupinus luteus L.) Rgulateurs naturels de la croissance vg tale. Proc. Fifth Intl. Conf. of Natural Plant Growth Reg, 363-375. Schneider, E.F. 1967. Conversion of the plant growth retardant (2-chloroethyl) trimethylammonium chloride to choline in s hoots of chrysanthemum and barley. Can. J. Biochem. 45:395-400. Sharma, N., S.R. Abrams, and D.R. Waterer. 2005. Uptake, movement, activity, and persistence of an abscisic analog (8 acetylene ABA methyl ester) in marigold and tomato. J. Plant Growth Reg. 24:28-35.

PAGE 59

59 Sharma, N., S.R. Abrams, D.R. Waterer. 2006. Ev aluation of abscisic acid analogs as holding agents for bedding plant seed lings. HortTechnology 16(1):71-77. Shindy, W.W., C.M. Asmundson, O.E. Smit h, and J. Kumamoto. 1973. Absorption and distribution of high sp ecific radioactivity 2-14C-abscisic acid in cotton seedlings. Plant Physiol. 52:443-447. Sondheimer, E., E.C. Galson, Y.P. Chang, and D.C. Walton. 1971. Asymmetry, its importance to the action and metabolism of abscisic acid. Science 174:829-831. Stamps, R. H., and A. L. Chandler. 2005. Effects of S-ABA on water lo ss and dessication of containerized Hibiscus rosa-sinensis HortScience 40(4): 1115-1116. Stephan, U., and H.R. Schutte. 1970. Zum meta bolismus von chlorcholin chlorid in hheren pflanzen. Biochem. Physiol. Pflanzen (BPP) 161:499 Sterrett, J.P.. 1985. Paclobutrazol: A promising growth inhibitor for injection into woody plants. J. Amer. Soc. Hort. Sci. 110(1):4-8. Sugavanam, B. 1984. Diastereoisomers and enantio mers of paclobutrazol: Their preparation and biological activity. Pesticide Sci. 15:296-302. Tal, M., and D. Imber. 1970. Abnormal stomatal behavior and hormonal imbalance in flacca, a wilty mutant of tomato. II. Auxinand abscisic acid-like activity. Plant Physiol. 46:373376. Tardieu, F., and W.J. Davies. 1993. Integration of hydraulic and chemical signaling in the control of stomatal conductan ce and water status of droughted plants. Plant, Cell, Environ. 16:341-349. Tolbert, N.E.. 1960a. (2-chloroethyl)trimethyl ammonium chloride and related compounds as plant growth substances. I. Chemical stru cture and bioassay. J. Biol. Chem. 235(2):475479. Tolbert, N.E.. 1960b. (2-chloroethyl)trimethyla mmonium chloride and related compounds as plant growth substances. II. Effect of growth of wheat. Plant Physiol. 35(3):380-385. Tolbert, N.E.. 1961. Alteration of plant growth by chemicals. Bul. Torrey Botanical Club 88(5):313-320. Tukey, H.B., F.W. Went, R.M. Muir, J. Van Overbeek. 1953. Nomenclature of chemical plant growth regulators: Report by a committee of the American Society of Plant Physiologists. Plant Physiol. 29:307-308. Wang, S.Y., T. Sun, and M. Faust. 1986. Tran slocation of paclobutr azol, a gibberellin biosynthesis inhibitor, in apple seedlings. Plant Physiol. 82:11-14.

PAGE 60

60 Walton, D.C. and E. Sondheimer. 1971. Metabolism of 2-14C-(RS)-abscisic acid in bean axes. Plant Physiol. 47(S):22-23. Walton, D.C., and Y. Li. 1995. Abscisic acid biosynthesis and metabolism, p. 140-157. In: P.J. Davies (ed.). Plant hormones: Physiology, biochemistry, and molecular biology. Kluwer Academic Publishers, Dordrecht, Neth. Wareing, P.F.. 1978. Abscisic acid as a natural gr owth regulator. Phil. Trans. Royal Soc. London B 284:483-498. Wright, S.T.C.. 1978. Phytohormones and stress phenomena, p. 495-536. In: D.S. Letham, P.B. Goodwin, and T.J.V. Higgins (eds.) P hytohormones and related compounds a comprehensive treatise, v. 2, Elsevier/North Holland Biomedical Press, Amsterdam, Neth.. Wright, S.T.C. and R.W.P. Hiron. 1969. (+)-Absci sic acid, the growth in hibitor induced in detached wheat leaves by a period of wilting. Nature 224:719-720. Wright, S.T.C., and R.W.P. Hiron. 1970. The accumu lation of abscisic acid in plants during wilting and under other stress conditions, p 291-298. In: DJ Carr (ed). Plant Growth Substances, Springer-Verlag, New York, NY. Zeevaart, J.A.D.. 1980. Changes in levels of absc isic acid and its metabo lites in excised leaf blades of Xanthium strumarium during and after water stre ss. Plant Physiol. 66:672-678. Zeevaart, J.A.D. and R.A. Creelman. 1988. Meta bolism and physiology of abscisic acid. Ann. Rev. Plant Physiol. Plant Mol. Biol. 39:439-473. Zhang, J. and W.J. Davies. 1987. Increased synthe sis of ABA in partially dehydrated root-tips and ABA transport from roots to l eaves. J. Expt. Bot. 38(197):2015-2023. Zhang, J., and W.J. Davies. 1991. Antitranspirant act ivity in xylem sap of maize plants. J. Expt. Bot. 42(236):317-321. Zhang, J., and W.J. Davies. 1989. Abscisic acid produced in dehydrating roots may enable the plant to measure the water status of the soil. Plant, Cell, Environ. 12:73-81. Zhang, J., and W.J. Davies. 1990. Changes in th e concentration of ABA in xylem sap as a function of changing soil water status can account for changes in leaf conductance and growth. Plant, Cell, and Environ. 13:277-285.

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61 BIOGRAPHICAL SKETCH Jessica Bold t was raised in Melbourne, Florida and became interested in horticulture through working in the family greenhouse operation. She attended the University of Florida and completed two internships during her degree program, a six-month internship at Whites Nursery and Greenhouse in Chesapeake, VA, and a three-m onth internship at Pleasant View Gardens in Loudon, NH. She graduated in 2005 with a B.S in environmental horticulture (landscape and nursery management specialization) and a B.A. in business administration Jessica was a graduate research assistant duri ng her graduate program at the University of Florida and conducted variety tria ling on pre-release plant materi al while assisting with the public trial garden. She graduated in 2008 with a M.S. in horticultural science (environmental horticulture specialization).


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